Sorbitol oxidase reagent from xanthomonas

ABSTRACT

The sorbitol oxidase of the present invention catalyzes the reaction in which D-sorbitol is oxidized D-glucose and hydrogen peroxide.

This application is a divisional of application Ser. No. 08/162,628,filed Dec. 7, 1993 now U.S. Pat. No. 5,472,862.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel enzyme, sorbitol oxidase, aprocess for producing the same, and the use thereof.

2. Description of the Related Art

Up to now, sorbitol oxidase, a novel enzyme, which catalyzes a reactionin which D-sorbitol is oxidized to D-glucose and hydrogen peroxide hasnot been found. Sorbitol is a compound which is produced in vivo fromglucose by aldose reductase. Because sorbitol has poor membranepermeability, the sorbitol has a tendency to be accumulated in a cellsuch as a red blood cell. The accumulation of sorbitol causes functionaldisorders such as the increase in an osmotic pressure in vivo, theswelling of a cell, etc. Each of the functional disorders is consideredas one of the diabetic complications. Thus, the content of sorbitol in ared blood cell derived from a body can be used for the diagnosis ofdiabetic complications.

Recently, a number of blocking agents for aldose reductase have beendeveloped as therapeutic agents for diabetic complications. Forconfirming the effects of these therapeutic agents, the measurement ofsorbitol in a red blood cell has been the subject of serious studiespaid much attention to.

A sorbitol dehydrogenase (L-Iditol: NAD⁺ 2-oxidoreductase (EC.1.1.1.14)), which catalyzes the following reaction, has been used as amethod for determining D-sorbitol.

    D-sorbitol+NAD.sup.+ ←D-fructose+NADH+H.sup.+

According to the above method, a sorbitol dehydrogenase is allowed toreact with D-sorbitol in a biological sample to produce NADH. Then, theNADH thus produced is measured, thereby determining the content ofD-sorbitol in the biological sample. However, this method has thefollowing problems.

Since the molecular extinction coefficient of NADH is small, thesensitivity of the measurement is low. In addition, for measuring theNADH with good sensitivity, a special apparatus is required. That is,the NADH is measured with good sensitivity by fluorometry at awavelength of 366 nm (excitation) and 452 nm (emission). Moreover, NADH,NAD⁺, etc., which are included in the biological sample may hinder thesensitivity of the measurement.

Another method for determining D-sorbitol, using the above-mentionedsorbitol dehydrogenase has been utilized, in which an artificialelectron receptor is used. This method is more useful than theabove-mentioned method for measuring NADH; however, the sensitivity forthe measurement in this method is not sufficiently precise for clinicaldiagnosis. Further, sorbitol dehydrogenase is a membrane-bound enzyme,so that it is unstable and tends to be inactivated.

In order to overcome the above-mentioned problems involved in thedetermination of D-sorbitol, attempts to develop another enzyme such asa sorbitol oxidase have been tried, but have not been found.

SUMMARY OF THE INVENTION

The inventors of the present invention earnestly studied the problemsinvolved in the determination of D-sorbitol using a conventionalsorbitol dehydrogenase; as a result, the inventors found a microorganismproducing a sorbitol oxidase from the soil of Sekigahara-cho, Fuwa-gun,Gifu Prefecture, Japan, thereby achieving the present invention.

The sorbitol oxidase of this invention catalyzes a reaction in whichD-sorbitol is oxidized to D-glucose and hydrogen peroxide.

In one embodiment of the present invention, the above-mentioned sorbitoloxidase has the following physico-chemical characteristics:

(1) Enzyme action: catalyzes the following reaction:

D-sorbitol+O₂ →D-glucose+H₂ O₂

(2) Substrate specificity:

specific activity for D-sorbitol, D-mannitol, D-xylitol, and D-arabitol.

(3) Optimum pH: 6.5 to 7.5, and

(4) Molecular weight: about 54,000 (gel filtration) about 43,000(SDS-PAGE)

According to another aspect of the present invention, a process forproducing a sorbitol oxidase comprises the steps of:

cultivating a sorbitol oxidase-producing microorganism which belongs tothe genus Xanthomonas in a nutrient culture;

producing the sorbitol oxidase; and

collecting the sorbitol oxidase.

In one embodiment of the present invention, the sorbitoloxidase-producing microorganism is Xanthomonas maltophilia TE3539.

According to another aspect of the present invention, a method fordetermining a polyol in a sample, comprises the steps of:

allowing the above-mentioned sorbitol oxidase to react with a samplecontaining at least one polyol selected from the group consisting ofD-sorbitol, D-mannitol, D-xylitol, and D-arabitol;

measuring a generated hydrogen peroxide produced by a reaction orD-glucose, D-mannose, D-xylose, or D-arabinose produced by the reaction,or oxygen consumed during the reaction;

thereby determining the polyol.

In one embodiment of the present invention, the sample comprises aliquid which is prepared from a food stuff or biological sample such asa red blood cell, especially which is prepared by treating a red bloodcell obtained by heparinized blood collection.

According to another aspect of the present invention, the reagent fordetermining D-sorbitol, includes a sorbitol oxidase which catalyzes thereaction in which D-sorbitol is oxidized to D-glucose and hydrogenperoxide.

In one embodiment of the present invention, the above-mentioned reagentfurther comprises 4-aminoantipyrin, phenol derivative or anilinderivative, and peroxidase.

According to another aspect of the present invention, a kit fordetermining D-sorbitol, comprises a sorbitol oxidase which catalyzes areaction in which D-sorbitol is oxidized to D-glucose and hydrogenperoxide.

In one embodiment of the present invention, the above-mentioned kitfurther comprises 4-aminoantipyrin, phenol derivative or anilinederivative, and peroxidase.

Thus, the invention described herein makes possible the advantages of(1) providing a novel sorbitol oxidase which catalyzes a reaction inwhich D-sorbitol is oxidized to D-glucose and hydrogen peroxide; (2)providing the determination of sorbitol in a sample with goodsensitivity, using the sorbitol oxidase; (3) providing the highlysensitive determination of sorbitol in a red blood cell, therebyaccurately diagnosing the diabetic complications; (4) providing areagent, and a kit for accurately diagnosing the complications ofdiabetes; and (5) providing a method for efficiently producing thesorbitol oxidase.

These and other advantages of the present invention will become apparentto those skilled in the art upon reading and understanding the followingdetailed description with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the relationship between the pH and therelative activity of a sorbitol oxidase of the present invention.

FIG. 2 is a graph showing the pH stability of the sorbitol oxidase ofthe present invention.

FIG. 3 is a graph showing the relationship between the reactiontemperature and the relative activity of the sorbitol oxidase of thepresent invention.

FIG. 4 is a graph showing the thermal stability of the sorbitol oxidaseof the present invention.

FIG. 5 is a graph showing an absorption spectrum of the sorbitol oxidaseof the present invention.

FIG. 6 is a standard curve showing the relationship between the amountof D-sorbitol and the absorbance at 500 nm by the use of the sorbitoloxidase of the present invention.

FIG. 7 is a graph showing the change of absorbance when D-sorbitol in ared blood cell is measured.

FIG. 8 is a standard curve showing the relationship between the amountof D-sorbitol and the absorbance at 340 nm by the use of a sorbitoldehydrogenase.

FIG. 9 is a graph showing the change of absorbance when D-sorbitol isdetermined by measuring the amount of produced glucose.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The novel enzyme of the present invention catalyzes the reaction inwhich D-sorbitol is oxidized to D-glucose and hydrogen peroxide.

The enzyme of the present invention has the following characteristics:

(1) Enzyme action catalyzes the following reaction:

    D-sorbitol+O.sub.2 →D-glucose+H.sub.2 O.sub.2

(2) Specific activity for D-sorbitol, D-mannitol, D-xylitol, andD-arabitol.

(3) Optimum pH: pH 6.5 to 7.5

(4) Molecular weight: 54,000 (gel filtration) 43,000 (SDS-PAGE)

(5) pH stability: pH 5 to 11

(6) Optimum temperature: 55° C.

(7) Thermostability: up to 50° C.

(8) Isoelectric point: pH 4.0

Any source (e.g., animals, plants, microorganisms, etc.) can be used forthe enzyme of the present invention, as long as a sorbitol oxidasehaving the above-mentioned characteristics can be produced from thesource. Preferably, the sorbitol oxidase-producing microorganismbelonging to the genus Xanthomonas is used, and the most preferably,Xanthomonas maltophilia TE3539 is used. Xanthomonas maltophilia TE3539is a strain isolated from the soil, and its mycological characteristicsare as follows:

(a) Morphological characteristics

(1) Shape: Short rods

(2) Size of a cell: 1.2×0.3 μm

(3) Pleomorphism of a cell: None

(4) Mobility: Yes, having a polar flagellum

(5) Sporulation: None

(b) Growth conditions on each medium

(1) Broth agar plate medium: After 48 hours of culture at 30° C., ayellow to creme-colored colony is formed. The periphery of the colony isentire and convex. The colony has a smooth, lustered and opaque surface.

(2) Bouillon culture: The strain grows satisfactorily, and the bouillonbecomes uniformly turbid.

(3) Gelatin stab culture: The strain grows satisfactorily, and only theupper portion thereof becomes a filiform. The gelatin is liquefied.

(4) Litmus milk: Color is not changed. Milk is solidified.

(c) Physiological characteristics

    ______________________________________                                        (1)  Gram staining:             Negative                                      (2)  Reduction of nitrate:      Negative                                      (3)  Denitrification:           Negative                                      (4)  MR test:                   Negative                                      (5)  VP test:                   Negative                                      (6)  Production of indole:      Negative                                      (7)  Production of hydrogen sulfide:                                                                          Negative                                      (8)  Hydrolysis of starch:      Negative                                      (9)  Use of citric acid:                                                           Koser's 19 medium:         Negative                                           Christensen's medium:      Positive                                      (10) Production of pigment: Water-soluble yellow                                   pigment is produced in a cell.                                           (11) Urease:                    Negative                                      (12) Oxidase:                   Negative                                      (13) Catalase:                  Positive                                      (14) β-galactosidase:      Positive                                      (15) Arginine dihydrolase:      Negative                                      (16) Lysine carboxylase:        Negative                                      (17) Ornithine carboxylase:     Negative                                      (18) Tryptophan deaminase:      Negative                                      (19) β-glucosidase:        Negative                                      (20) Protease:                  Positive                                      (21) Growth conditions:                                                            Growth temperature                                                            20° C.              Positive                                           30° C.              Positive                                           37° C.              Negative                                           40° C.              Negative                                           Growth pH                                                                     pH 4                       Negative                                           pH 7                       Positive                                           pH 9                       Positive                                      (22) Behavior toward oxygen: Aerobic                                          (23) O-F test (Hugh Leifson method):                                                                          Negative                                           (No degradation of sugar)                                                (24) Production of acid and gas from sugar                                                           Acid     Gas                                           ______________________________________                                        L-arabinose            Negative Negative                                      D-xylose               Negative Negative                                      D-glucose              Negative Negative                                      D-mannose              Positive Negative                                      D-fructose             Positive Negative                                      D-galactose            Negative Negative                                      Maltose                Positive Negative                                      Sucrose                Negative Negative                                      Lactose                Negative Negative                                      Trehalose              Negative Negative                                      D-sorbitol             Negative Negative                                      D-mannitol             Negative Negative                                      Inositol               Negative Negative                                      Giycerin               Positive Negative                                      Starch                 Negative Negative                                      Rhamnose               Negative Negative                                      D-melibiose            Negative Negative                                      D-amygdalin            Negative Negative                                      (25) Utilization of organic compound                                               D-glucose                  Positive                                           L-arabinose                Positive                                           D-mannose                  Positive                                           D-mannitol                 Positive                                           D-sorbitol                 Positive                                           N-acetyl-D-glucosamine     Negative                                           Maltose                    Positive                                           Potassium gluconate        Positive                                           n-caprinate                Negative                                           Adipic acid                Negative                                           dl-malic acid              Positive                                           Sodium citrate             Positive                                           Pbenyl acetate             Negative                                      ______________________________________                                    

An experiment for identifying the above-mentioned mycologicalcharacteristics was conducted in accordance with T. Hasegawa (1985),Revised edition "Classification and identification of microorganisms",Academy publishing center. The classification and identification ofmicroorganisms are based on "Bergey's Manual of Systematic Bacteriology"(1984).

From the literature and mycological characteristics, the above-mentionedstrain is decided to belong to the genus Xanthomonas. The strain sharesa number of characteristics with Xanthomonas campestris or Xanthomonasmaltophilia. The strain is not recognized to have plant pathogenicity.The strain is recognized to exist singly or in pairs. Moreover, thestrain does not produce acid from glucose but produces acid frommaltose. Thus, the strain is named as Xanthomonas maltophilia TE3539.The strain has been deposited as Xanthomonas maltophilia TE3539 in theNational Institute of Biosclerics and Human Technology with theaccession No. FERM BP-4512.

The enzyme of the present invention can be produced by cultivating theabove-mentioned strain, which produces a sorbitol oxidase, in a nutrientmedium, and isolating the sorbitol oxidase from the culture thusobtained. Any media such as a synthetic medium and a natural medium canbe used for cultivating the strain producing the sorbitol oxidase, aslong as the medium contains a carbon source, a nitrogen source, aninorganic substance, and other required nutrients which themicroorganism can assimilate. Examples of the carbon source includeglucose, glycerol, and the like. Examples of the nitrogen source includenatural substances containing nitrogen such as peptones, a broth, ayeast extract, and the like; and compounds containing inorganic nitrogensuch as ammonium chloride, ammonium citrate, and the like. Examples ofthe inorganic substances include potassium phosphate, sodium phosphate,magnesium sulfate, and the like. It is desired that sorbitol is added tothe medium as an inducer for producing the sorbitol oxidase.

In general, a microorganism is cultivated by shaking or by an aerationculture. Culture conditions vary depending upon the kind ofmicroorganism. Generally, the culture temperature is in the range of 20°to 35° C., preferably 25° to 30° C.; and the culture pH is in the rangeof 5 to 9, preferably 6 to 8. The culture period of a microorganism isgenerally one to seven days. The sorbitol oxidase is produced to beaccumulated in the cultured strain.

The enzyme of the present invention can be purified by a conventionalmethod. The enzyme is extracted by a disruption method using ultrasonicwaves; a mechanical disruption method using a glass beads, or Frenchpress; a method using a surfactant; or the like. Moreover, the extractthus obtained is treated by using an ammonium sulfate, sodium sulfate,etc.; a metal agglutination method using magnesium chloride, calciumchloride, etc.; an agglutination method using protamine,polyethyleneimine, etc.; an ion exchange chromatography methods usingresins such as diethylaminoethyl (DEAE)-sephalose, carboxymethyl(CM)-sephalose, etc.; or the like. Accordingly, the enzyme is purified.A crude enzyme solution or purified enzyme solution can be dried orpowdered by spray-drying, lyophilization, or the like. Alternatively,the crude enzyme solution or the purified enzyme solution is immobilizedon a suitable carrier to be used as immobilized enzyme. A knownimmobilization method can be employed.

Hereinafter, a method for determining the activity of the sorbitoloxidase of the present invention will be described. The activity of thesorbitol oxidase is determined by measuring H₂ O₂ produced by the enzymereaction.

First, the following reaction mixture is prepared.

Substrate solution (0.2M sorbitol in 0.1M potassium-phosphate buffer (pH7.0)) 50 ml

0.1 w/v % aqueous solution of 4-aminoantipyrin 10 ml

0.1 w/v % aqueous solution of phenol 20 ml 0.025 w/v % aqueous solutionof peroxidase (manufactured by Toyo Boseki Kabushiki Kaisha) 20 ml

Then, 3.0 ml of the reaction mixture is put in a cuvette and prewarmedat 37° C. for about 5 minutes. To this reaction mixture, 0.05 ml ofenzyme solution was added and allowed to react. The absorbance of thereaction mixture at 500 nm is recorded for 3 to 4 minutes by using aspectrophotometer set at 37° C. From the initial linear portion of theabsorbance curve thus obtained, the change of absorbance per minute isobtained. As a control, 0.1M potassium-phosphate buffer (pH 7.0) isadded instead of the enzyme solution, and the same process is conducted.The one unit (U) of sorbitol oxidase activity is defined as oxidizing 1μmole of sorbitol per minute under the above conditions.

Further, according to the present invention the amount of polyol can bedetermined. The sorbitol oxidase having the above-mentionedcharacteristics is allowed to react with a sample containing at leastone polyol selected from the group consisting of D-sorbitol, D-mannitol,D-xylitol, and D-arabitol. Then, hydrogen peroxide thus produced orD-glucose, D-mannose, D-xylose, or D-arabinose thus produced, or oxygenthus consumed is measured, thereby determining the amount of the polyol.

Examples of the sample containing the polyol such as D-sorbitol,D-mannitol, D-xylitol, D-arabitol, etc., include food and biologicalsamples such as a red blood cell.

Hydrogen peroxide produced by the sorbitol oxidase can be measured byusing a hydrogen peroxide electrode, a chromogenic method, a fluorescentmethod, etc. An example of the chromogenic method includes a methodusing chromogenic agents, such as 4-aminoantipyrin, a phenol derivative,an aniline derivative, benzothiazolinone, etc., to develop color byreacting with peroxidase. An example of the fluorescent method includesa method using an acridinium ester, lumigenine, or derivatives thereof,luminol or a derivative thereof.

D-glucose produced by the sorbitol oxidase can be measured by anultraviolet absorbance spectrum, using glucose dehydrogenase orhexokinase/glucose-6-dehydrogenase phosphate. Alternatively, theD-glucose is measured by a method using a formazan reaction utilizingdiaphorase. D-mannose, D-xylose, and D-arabinose can be measured by aknown method.

Oxygen consumed by the sorbitol oxidase can be measured by using aWarburg's manometer, an oxygen electrode, etc.

According to the present invention, the sorbitol oxidase of the presentinvention is reacted with a sample containing the above-mentioned polyolunder the following conditions, and then a product produced by thereaction or oxygen consumed is measured.

A reagent containing the enzyme of the present invention is allowed toreact with a sample for 5 to 15 minutes under the conditions of pH 5 to9, preferably pH 6 to 8, a temperature of not more than 50° C.,preferably in the range of 25° to 40° C. As a buffer to be used, aphosphate buffer, a Tris-HCl buffer, borate buffer, and a GOOD's bufferare preferred.

According to the present invention, the polyol can be measured by a Ratemethod or End method.

According to the present invention, a reagent or a kit containing asorbitol oxidase is used for measuring sorbitol in a red blood cell. Thereagent or kit can contain an agent required for measuring hydrogenperoxide or D-glucose produced by the reaction of the sorbitol oxidase.In the case where hydrogen peroxide is measured, the reagent or kit cancontain the above-mentioned chromogenic agent and peroxidase. In thecase where D-glucose is measured, the reagent or kit can contain glucosedehydrogenase or hexokinase/glucose-6-dehydrogenase phosphate.

The novel enzyme of the present invention reacts with D-sorbitol toproduce D-glucose and hydrogen peroxide. By using this enzyme, polyolssuch as D-sorbitol, D-mannitol, D-xylitol, D-arabitol, etc. can beefficiently measured. In particular, D-sorbitol is a cause of thediabetic complications, so that a method for measuring D-sorbitol withgood sensitivity has been desired for a long time. The use of thesorbitol oxidase of the present invention makes it possible to diagnosethe complications of diabetes with good sensitivity.

Hereinafter, the present invention will be described by way ofillustrative examples with reference to the drawings.

EXAMPLE 1

First, 100 ml of medium (pH 7.0) containing 1 w/v % sorbitol, 1 w/v %polypsprone, 0.5 w/v % yeast extract, and 0.5 w/v % NaCl in a 500 mlSakaguchi flask was sterilized in an autoclave at 121° C. for 15minutes. A loop of Xanthomonas maltophilia TE3539 (FERM BP-4512) wasinoculated as a seed and cultivated at 30° C. for 48 hr to obtain aseed. Then, 6 l of the above medium in a 10 l jar fermentor wassterilized at 121° C. for 15 minutes, and was cooled. Thereafter, 100 mlof the seed culture was transferred to this medium and cultivated for 2days under the conditions of 300 rpm, an aeration rate of 2 l/min., anda temperature of 30° C. The culture thus obtained was centrifuged tocollect cells. These cells were suspended in 50 mM phosphate buffer (pH7.0).

The cells thus collected were disrupted by a French press, followed bybeing centrifuged to obtain a supernatant. The crude enzyme solutionthus obtained was purified to a specific activity of 13 U/mg by gelfiltration, using the fractionation with ammonium sulfate,DEAE-Sepharose chromatography, phenyl-Sepharose chromatography, andSephadex G-200.

The sorbitol oxidase thus obtained had the following characteristics:

(1) Catalyzes the following reaction

    D-sorbitol+O.sub.2 →D-glucose+H.sub.2 O.sub.2

(2) Substrate specificity

The enzyme activity was determined in the case where variousmonosaccharides and derivatives thereof were used as a substrate. Thevalue of enzyme activity with respect to D-sorbitol was set at 100.

                  TABLE 1                                                         ______________________________________                                        Substrate         Relative activity (%)                                       ______________________________________                                        D-sorbitol        100.00                                                      D-mannitol        86.60                                                       Inositol          0.40                                                        D-xylitol         89.90                                                       D-arabitol        23.20                                                       Glycerol          3.50                                                        D-glucose         0.54                                                        D-fructose        2.88                                                        D-galactose       0.42                                                        D-mannose         1.01                                                        L-sorbose         10.60                                                       Maltose           0.00                                                        D-glucosamine hydrochloride                                                                     0.00                                                        ______________________________________                                    

(3) Km value

The Km value with respect to sorbitol was 0.60 mM.

(4) Optimum pH

The enzyme activity was determined in 50 mM potassium-phosphate buffer(pH 5.5 to 8.0) and 50 mM Tris-HCl buffer (pH 7.5 to 8.5). The resultsare shown in FIG. 1. The optimum pH was 6.5 to 7.5.

(5) pH stability

The sorbitol oxidase was kept in a Britton-Robinson's buffer (pH 3 to12) at 25° C. for 24 hours to determine residual activity thereof. Theresults are shown in FIG. 2. The stable pH is 5 to 11.

(6) Optimum temperature

The enzyme activity at each temperature was determined. The results areshown in FIG. 3. The optimum temperature was 55° C.

(7) Thermostability

The enzyme of the present invention was kept in 50 mMpotassium-phosphate buffer (pH 8.0) for 15 minutes. Thereafter, theresidual enzyme activity was determined. The results are shown in FIG.4. The residual enzyme activity was stable up to 60° C.

(8) Molecular weight: 54,000 (gel filtration) 43,000 (SDS-PAGE)

(9) Isoelectric point: pH 4.0 (isoelectric focusing)

(10) Absorption spectrum

The purified enzyme had a sharp peak at 273 nm and broad peaks at 350 nmand 450 nm, thus showing a typical pattern of flavin enzyme (FIG. 5).

EXAMPLE 2

As a reaction solution, a reaction reagent containing 50 mM phosphatebuffer (pH 7.0), 0.50 mM 4-aminoantipyrin, 2.0 mM phenol, 4.5 U/ml ofperoxidase (manufactured by Toyo Boseki Kabushiki Kaisha), and 1 U/ml ofenzyme of the present invention Was prepared. Then, 0.05 ml each ofsample solutions (6 kinds) was respectively added to 1 ml of thereaction reagent this case, 6 kinds of sample solutions contained 0,0.1, 0.2, 0.3, 0.4, and 0.5 μmole of D-sorbitol, respectively. Eachmixture (i.e., 0.05 ml of the sample solution and 1 ml of the reactionreagent) was allowed to react at 37° C. for 5 minutes, and theabsorbance of each mixture was measured at 500 nm by aspectrophotometer. The results are shown in FIG. 6. As is understoodfrom this figure, the relationship between the absorbance and the amountof D-sorbitol (i.e., standard curve) was expressed as a straight line inthe range of 0 to 0.5 μmole of D-sorbitol. Thus, in this range, it waspossible to measure D-sorbitol.

EXAMPLE 3

From blood obtained by heparinized blood collection, a 2 ml packed cellof red blood cells was prepared. Then, 3 ml of cooled (4%) perchloricacid was added to the packed cell to remove proteins. To the supernatantthus obtained, 0.25 ml of 2.5M potassium carbonate was added, mixed andallowed to stand at 4° C. for 2 hours. Then, the mixture was centrifugedto obtain a supernatant as a sample. Then, 0.05 ml of the sample wasmixed with 1 ml of the reaction reagent prepared in Example 2. Themixture thus obtained was allowed to react at 37° C., FIG. 7 shows thechange of the absorbance of the mixture at 500 nm with time.

Comparative Example 2

As a reaction solution, a reaction reagent containing Tris-HCl buffer(pH 8.0), 4 mM NAD⁺, and 5 U/ml of sorbitol dehydrogenase (manufacturedby Boehringer) was prepared. Then, 0.05 ml each of sample solutions (6kinds) was respectively added to 1 ml of the reaction reagent. In thiscase, the 6 kinds of sample solutions contained 0, 0.1, 0.2, 0.3, 0.4,and 0.5 μmole of D-sorbitol. Each mixture (i.e., 0.05 ml of the samplesolution and 1 ml of the reaction reagent) was allowed to react at 37°C. for 30 minutes, and the absorbance of each mixture was measured at340 nm by a spectrophotometer. The results are shown in FIG. 8. Thismeasurement was poor in sensitivity, and a calibration curve did nothave linearity sufficient for measurement.

EXAMPLE 4

First, 0.05 ml each of sample solutions (3 kinds) was added to 1 ml ofthe reaction reagent prepared in Example 2. In this case, the 3 kinds ofsample solutions contained 0.1 μmole of D-mannitol, 0.1 μmole ofD-xylitol, and 0.1 μmole of D-arabitol, respectively. Each mixture(i.e., 0.05 ml of the sample solution and 1 ml of the reaction reagent)was allowed to react at 37° C. for 5 minutes, and the absorbance of eachmixture was measured by a spectrophotometer at 500 nm. The resultsshowed that the respective absorbances were 0.042 (D-mannitol), 0.044(D-xylitol), and 0.040 (D-arabitol). Thus, these absorbances were almostthe same as that of D-sorbitol, i.e., 0.042.

EXAMPLE 5

A reaction reagent containing 50 mM Tris-HCl buffer (pH 8.0), 4 mM NAD⁺,30 U/ml of glucose dehydrogenase (manufactured by Toyo Boseki KabushikiKaisha), and 1 U/ml of the enzyme of the present invention was prepared.Then, 0.05 ml of sample solution containing 0.5 μmole of D-sorbitol wasadded to the reaction reagent. The mixture thus obtained was allowed toreact, and the absorbance of the mixture was obtained at 340 nm by aspectrophotometer. Due to the reaction of the enzyme of the presentinvention, D-glucose was produced from D-sorbitol. The present exampleshowed that D-sorbitol could be measured by the amount of D-glucoseproduced.

Various other modifications will be apparent to and can be readily madeby those skilled in the art without departing from the scope and spiritof this invention. Accordingly, it is not intended that the scope of theclaims appended hereto be limited to the description as set forthherein, but rather that the claims be broadly construed.

What is claimed is:
 1. A reagent for determining sorbitol, whichcomprises sorbitol oxidase that catalyzes the following reaction:sorbitol+O₂ →glucose+and hydrogen peroxide, wherein the sorbitol oxidaseis isolated from Xanthomonas maltophilia and wherein said sorbitoloxidase further has the following properties:(1) substrate specificity:specific activity for D-sorbitol, D-mannitol, D-xylitol, and D-arabitol;(2) optimum pH: 6.5 to 7.5; and (3) molecular weight: about 54,000 asdetermined by gel filtration or about 43,000 as determined by SDS-PAGE.2. The reagent of claim 1, wherein the sorbitol oxidase is isolated fromXanthomonas maltophilia FERM BP-4512.
 3. A kit for determiningD-sorbitol, which comprises sorbitol oxidase that catalyzes thefollowing reaction: sorbitol+O₂ →glucose+H₂ O₂ and is isolated fromXanthomonas maltophilia, and wherein said sorbitol oxidase further hasthe following properties:(1) substrate specificity: specific activityfor D-sorbitol, D-mannitol, D-xylitol, and D-arabitol; (2) optimum pH:6.5 to 7.5; and (3) molecular weight: about 54,000 as determined by gelfiltration or about 43,000 as determined by SDS-PAGE.
 4. A kit of claim3, wherein said sorbitol oxidase is isolated from Xanthomonasmaltophilia FERM BP-4512.
 5. The reagent of claim 1, which furthercomprises 4-aminoantipyrin, phenol derivatives or aniline derivative andperoxidase.
 6. The kit of claim 3, which further comprisesaminoantipyrin, phenol derivatives or aniline derivative and peroxidase.